Monoclonal antibodies and uses thereof

ABSTRACT

The technology relates to monoclonal antibodies useful in the identification of cancer cells. In one embodiment, mAbs with specificity for tumor antigens are provided. In one embodiment, methods for treating cancer using mAbs are provided. In another embodiment, methods for detecting cancerous cells are provided. In another embodiment, kits for detecting cancerous cells are provided.

RELATED APPLICATIONS

This application is a continuation application of U.S. Ser. No.12/836,299 filed Jul. 14, 2010, now U.S. Pat. No. 8,361,706 which is adivisional application of U.S. Ser. No. 11/704,031 filed Feb. 8, 2007,now U.S. Pat. No. 7,790,857 B2, which claims priority to U.S. Ser. No.60/772,146 filed Feb. 10, 2006.

FIELD OF THE INVENTION

The technology relates to monoclonal antibodies useful in theidentification of cancer cells.

BACKGROUND OF THE INVENTION

Monoclonal antibodies have proven useful in the diagnosis and treatmentof diseases such as cancer. Those active in the field of diseaseprevention and diagnosis understand that for certain conditions,additional reagents are required to improve the prevention and/ortreatment of such diseases. One such area is oncology. Despite recentadvances in the field, there is a need for additional highly specificdiagnostic reagents useful in diagnosing cancer. The technologydescribed herein provides such reagents, as shown below.

SUMMARY OF THE INVENTION

The technology relates to monoclonal antibodies useful in theidentification of cancer cells. In one embodiment, mAbs with specificityfor tumor antigens are provided. In one embodiment, methods for treatingcancer using mAbs are provided. In another embodiment, methods fordetecting cancerous cells are provided. In another embodiment, kits fordetecting cancerous cells are provided.

DETAILED DESCRIPTION

The technology described herein relates to antibodies for use inidentifying cells expressing the BFA4 protein as well as diagnosing,treating and/or preventing one or more diseases associated with theexistence of such cells. The tem “antibody” or “antibodies” includeswhole or fragmented antibodies in unpurified or partially purified form(i.e., hybridoma supernatant, ascites, polyclonal antisera) or inpurified form. A “purified” antibody is one that is separated from atleast about 50% of the proteins with which it is initially found (i.e.,as part of a hybridoma supernatant or ascites preparation). Preferably,a purified antibody is separated from at least about 60%, 75%, 90%, or95% of the proteins with which it is initially found. Suitablederivatives may include fragments (i.e., Fab, Fab₂ or single chainantibodies (Fv for example)), as are known in the art. The antibodiesmay be of any suitable origin or form including, for example, murine(i.e., produced by murine hybridoma cells), or expressed as humanizedantibodies, chimeric antibodies, human antibodies, and the like.

Methods of preparing and utilizing various types of antibodies arewell-known to those of skill in the art and would be suitable inpracticing the present invention (see, for example, Harlow, et al.Antibodies: A Laboratory Manual, Cold Spring Harbor Laboratory, 1988;Harlow, et al. Using Antibodies: A Laboratory Manual, Portable ProtocolNo. 1, 1998; Kohler and Milstein, Nature, 256:495 (1975)); Jones et al.Nature, 321:522-525 (1986); Riechmann et al. Nature, 332:323-329 (1988);Presta (Curr. Op. Struct. Biol., 2:593-596 (1992); Verhoeyen et al.(Science, 239:1534-1536 (1988); Hoogenboom et al., J. Mol. Biol.,227:381 (1991); Marks et al., J. Mol. Biol., 222:581 (1991); Cole etal., Monoclonal Antibodies and Cancer Therapy, Alan R. Liss, p. 77(1985); Boerner et al., J. Immunol., 147 (1):86-95 (1991); Marks et al.,Bio/Technology 10, 779-783 (1992); Lonberg et al., Nature 368 856-859(1994); Morrison, Nature 368 812-13 (1994); Fishwild et al., NatureBiotechnology 14, 845-51 (1996); Neuberger, Nature Biotechnology 14, 826(1996); Lonberg and Huszar, Intern. Rev. Immunol. 13 65-93 (1995); aswell as U.S. Pat. Nos. 4,816,567; 5,545,807; 5,545,806; 5,569,825;5,625,126; 5,633,425; and, 5,661,016). In certain applications, theantibodies may be contained within hybridoma supernatant or ascites andutilized either directly as such or following concentration usingstandard techniques. In other applications, the antibodies may befurther purified using, for example, salt fractionation and ion exchangechromatography, or affinity chromatography using Protein A, Protein G,Protein A/G, and/or Protein L ligands covalently coupled to a solidsupport such as agarose beads, or combinations of these techniques. Theantibodies may be stored in any suitable format, including as a frozenpreparation (i.e., −20° C. or −70° C.), in lyophilized form, or undernormal refrigeration conditions (i.e., 4° C.). When stored in liquidform, it is preferred that a suitable buffer such as Tris-bufferedsaline (TBS) or phosphate buffered saline (PBS) is utilized.

To identify potential antigens useful in producing monoclonal antibodiesreactive against cancer cells, differential gene expression profiling oftumor and normal tissues was performed using DNA microarrays, followedby validation of gene expression at the protein level and elucidation ofT-cell reactivity against predicted peptide epitopes. Gene profilingusing microarrays was performed with 54 breast tumor specimens obtainedimmediately following surgery, including ductal carcinoma in situ,invasive ductal carcinoma, and liver and lung metastases. In each case,the gene expression patterns in the tumours were compared to expressionprofiles obtained from over 100 normal tissues and cell types in orderto identify genes over-expressed specifically in breast cancer. Thisscreening approach led to the detection of a number of over-expressed,breast tumour-specific genes. One of the most highly over-expressedgenes (“BFA4”) was found to be identical to a GATA familytranscriptional regulator shown to associate with a rare autosomaldominant genetic disorder called trichorhinophalangeal syndrome type 1(TRPS-1) (Momeni, P. et al. Nat Genet 24, 71-4 (2000); Malik, et al. MolCell Biol 22, 8592-600 (2002); Ludecke, et al. Am J Hum Genet 68, 81-91(2001); Kaiser, et al. Eur J Hum Genet 12, 121-6 (2004)). BFA4 was foundto be expressed in greater than 90% of breast cancers at the proteinlevel. Antibodies reactive therewith, and in particular monoclonalantibodies (mAbs) reactive therewith, are described herein.

One such mAb is termed “8D11”, and a murine hybridoma producing 8D11 wasdeposited on Jul. 12, 2005 with the American Type Culture Collection(ATCC), 10801 University Blvd., Manassas, Va. 20110-2209, U.S.A. underthe provisions of the Budapest Treaty for the International Recognitionof the Deposit of Microorganism for the Purposes of Patent Procedure,and accorded Patent Deposit Designation PTA-6852. A hybridoma producinga second antibody showing reactivity with the BFA4 protein is termed“8A1” was similarly deposited with the ATCC on Jul. 12, 2005 andaccorded Patent Deposit Designation PTA-6853. Other antibodies,including polyclonal antisera or preparations of isolated antibodiescontained therein, for example, are also provided herein.

In certain embodiments, preparations of 8D11 and/or 8A1 are provided.Such preparations may include unpurified antibody as found in ahybridoma supernatants or ascites preparation, partially purifiedpreparations, or purified preparations. Thus, provided herein areantibody preparations containing 8D11 and/or 8A1 purified to about 50%,60%, 75%, 90%, or 95% purity. Typically, such preparations include abuffer such as phosphate- or tris-buffered saline (PBS or TRIS,respectively). Also provided are derivatives of 8D11 and/or 8A1including fragments (Fab, Fab₂ or single chain antibodies (Fv forexample)), humanized antibodies, chimeric antibodies, human antibodies,and the like. The genes encoding the variable and hypervariable segmentsof 8D11 and/or 8A1 may also be isolated from the hybridomas expressingthe same cloned into expression vectors to produce certain antibodypreparations (i.e., humanized antibodies). Methods for producing suchpreparations are well-known in the art.

The skilled artisan has many suitable techniques for using theantibodies described herein to identify biological samples containingproteins that bind thereto. For instance, the antibodies may be utilizedto isolate BFA4 protein using, for example, immunoprecipitation or othercapture-type assay. This well-known technique is performed by attachingthe antibody to a solid support or chromatographic material (i.e., abead coated with Protein A, Protein G and/or Protein L). The boundantibody is then introduced into a solution either containing orbelieved to contain the BFA4 protein. BFA4 protein then binds to theantibody and non-binding materials are washed away under conditions inwhich the BFA4 protein remains bound to the antibody. The bound proteinmay then be separated from the antibody and analyzed as desired. Similarmethods for isolating a protein using an antibody are well-known in theart.

The antibodies may also be utilized to detect BFA4 protein within abiological sample. For instance, the antibodies may be used in assayssuch as, for example, flow cytometric analysis, ELISA, immunoblotting(i.e., western blot), in situ detection, immunocytochemistry, and/orimmunhistochemistry. Methods of carrying out such assays are well-knownin the art.

To assist the skilled artisan in using 8D11 and 8A1, the same may beprovided in kit format. A kit including 8D11 and/or 8A1 and optionallyother components necessary for using the antibodies to detect cellsexpressing BFA4 is provided. The antibodies of the kit may be providedin any suitable form, including frozen, lyophilized, or in apharmaceutically acceptable buffer such as TBS or PBS. The kit may alsoinclude other reagents required for utilization of the antibodies invitro or in vivo such as buffers (i.e., TBS, PBS), blocking agents(solutions including nonfat dry milk, normal sera, Tween-20 Detergent,BSA, or casein), and/or detection reagents (i.e., goat anti-mouse IgGbiotin, streptavidin-HRP conjugates, allophycocyanin, B-phycoerythrin,R-phycoerythrin, peroxidase, fluors (i.e., DyLight, Cy3, Cy5, FITC,HiLyte Fluor 555, HiLyte Fluor 647), and/or staining kits (i.e., ABCStaining Kit, Pierce)). The kits may also include other reagents and/orinstructions for using the antibodies in commonly utilized assaysdescribed above such as, for example, flow cytometric analysis, ELISA,immunoblotting (i.e., western blot), in situ detection,immunocytochemistry, immunhistochemistry.

In one embodiment, the kit provides 8D11 and/or 8A1 in purified form. Inanother embodiment, 8D11 and/or 8A1 are provided in biotinylated formeither alone or along with an avidin-conjugated detection reagent (i.e.,antibody). In another embodiment, the kit includes a fluorescentlylabelled 8D11 and/or 8A1 which may be used to directly detect BFA4protein. Buffers and the like required for using any of these systemsare well-known in the art and may be prepared by the end-user orprovided as a component of the kit. The kit may also include a solidsupport containing positive- and negative-control protein and/ortissue-samples. For example, kits for performing spotting or westernblot-type assays may include control cell or tissue lysates for use inSDS-PAGE or nylon or other membranes containing pre-fixed controlsamples with additional space for experimental samples. Kits forvisualization of BFA4 in cells on slides may include pre-formattedslides containing control cell or tissue samples with additional spacefor experimental samples.

8D11 and/or 8A1 and/or derivatives thereof may also be incorporated intocompositions of the invention for use in vitro or in vivo. Theantibodies or derivatives thereof may also be conjugated to functionalmoieties such as cytotoxic drugs or toxins, or active fragments thereofsuch as diphtheria A chain, exotoxin A chain, ricin A chain, abrin Achain, curcin, crotin, phenomycin, enomycin, among others. Functionalmoieties may also include radiochemicals.

8D11 and 8A1 and/or derivatives thereof may be used in assays todetermine the presence of a disease state in a patient, to predictprognosis, or to determine the effectiveness of a chemotherapeutic orother treatment regimen. Expression profile assays, performed asdescribed herein or as is otherwise known in the art, may be used todetermine the relative level of expression of BFA4. The level ofexpression may then be correlated with base (i.e., control) levels todetermine whether a particular disease is present within the patient,the patient's prognosis, or whether a particular treatment regimen iseffective. For example, if the patient is being treated with aparticular chemotherapeutic regimen, a decreased level of expression ofan immunogenic target in the patient's tissues (i.e., in peripheralblood, breast tissue biopsy) may indicate the regimen is decreasing thecancer load in that host. Similarly, if the level of expression isincreasing, this may indicate the regimen is not having the desiredeffect and another therapeutic modality may be selected.

It is also possible to use the antibodies described herein as reagentsin drug screening assays. The reagents may be used to ascertain theeffect of a drug candidate on the expression of the immunogenic targetin a cell line, or a cell or tissue of a patient. The expressionprofiling technique may be combined with high throughput screeningtechniques to allow rapid identification of useful compounds and monitorthe effectiveness of treatment with a drug candidate (see, for example,Zlokamik, et al., Science 279, 84-8 (1998)). Drug candidates may bechemical compounds, nucleic acids, proteins, antibodies, or derivativestherefrom, whether naturally occurring or synthetically derived. Drugcandidates thus identified may be utilized, among other uses, aspharmaceutical compositions for administration to patients or for use infurther screening assays.

The antibodies described herein may be prepared as injectablepreparation, such as in suspension in a non-toxic parenterallyacceptable diluent or solvent. Suitable vehicles and solvents that maybe utilized include water, Ringer's solution, and isotonic sodiumchloride solution, TBS and PBS, among others. In certain applications,the antibodies are suitable for use in vitro. In other applications, theantibodies are suitable for use in vivo. The preparations suitable foruse in either case are well-known in the art and will vary depending onthe particular application.

All references cited herein are hereby incorporated by reference. Abetter understanding of the present invention and of its many advantageswill be had from the following examples, given by way of illustration.

EXAMPLES Example 1 Generation and Characterization of Polyclonal andMonoclonal Antibodies

A. Polyclonal Antibodies

Polyclonal antibodies were generated against 22-mer or 23-mer peptidesconjugated to keyhole limpet hemocyanin (KLH). The peptide sequenceswere as follows:

(CLP2589; SEQ ID NO: 1) KLH-MVRKKNPPLRNVASEGEGQILE,(CLP2590; SEQ ID NO: 2) KLH-SPKATEETGQAQSGQANCQGLS,(CLP25691; SEQ ID NO: 3) KLH-VAKPSEKNSNKSIPALQSSDSG,(CLP2592; SEQ ID NO: 4) KLH-NHLQGSDGQQ SVKESKEHSCTK,(CLP2593; SEQ ID NO: 5) KLH-NGEQIIRRRTRKRLNPEALQAE, and(CLP2594; SEQ ID NO: 6) KLH-ANGASKEKTKAPPNVKNEGPLNV.

White New Zealand rabbits (Harlan) were immunized intramuscularly with100 μg of peptide emulsified in CFA and boosted on days 15, and 28 withpeptide and IFA. Final antibody titres from bleeds taken on day 57ranged from 1:40,000 to 1:320,000. Polyclonal antibodies (pAb) weregenerated against a series of 22-mer peptides in the BFA4 sequence. Oneof these pAb, designated 2589, recognized a specific band in BT474 andMDA-MB 453 breast tumour cells migrating at 200-220 kDa followingSDS-PAGE and Western blotting. The BFA4 protein also migrated with thesame molecular weight in lysates from NYVAC-BFA4-infected Vero cells(data not shown).

B. Monoclonal Antibodies

Monoclonal antibodies (mAbs) were generated against a 54 kDa His-taggedNH₂ amino terminal recombinant protein of BFA4 (BFA4-N54). Therecombinant protein was cloned into pGEX4T1-6His is plasmid, expressedin E. coli, and purified from bacterial lysates by column chromatographyon NiNTA columns. Monoclonal antibodies were produced at ImmunoPreciseAntibodies Ltd. (Victoria, BC, Canada). Briefly, BFA4-N54 protein (50μg) was emulsified in CFA and injected subcutaneously into Balb/c mice.Hybridomas were generated after 3 boosts and screened in ELISA. Out of21 clones, two clones (designated 8D11 and 8A1) were found to exhibitoptimal ELISA and immunoblot reactivity against recombinant therecombinant protein and in lysates of BFA4-expressing cell lines.Hybridoma culture supernatants were purified using Protein Gchromatography. The migration of the BFA4 protein in SDS-PAGE wasfurther confirmed using immunoblot analysis with two monoclonalantibodies (mAb) designated 8D11 and 8A1. A single 200-220 kDa band wasfound in BT474 and MDA-MB453 cell lysates with no detectable expressionin COS cells used as a specificity control.

To further confirm the specificity of the mAbs, breast cancer cell lines(BT474, MDA-MB 453, and MDA-MB-231) and control cell lines (COS andHeLa) were grown on glass coverslips in 24-well plates and washed withPBS. The cells were then fixed with 3% paraformaldehyde in PBS, washedthree times with PBS, 10 mM NH₄Cl and permeabilized in PBS, 0.1% saponin(PBS/sapo) for 10 min. The cells were stained with primary antibodiesfollowed by Alexa-488-coupled secondary antibodies diluted inPBS/sapo/HS. The cells were counterstained with 0.05% Evans blue.Stained slides were analyzed with a Zeiss Axioplan epifluorescencemicroscope or a Zeiss LSM confocal fluorescence microscope equipped withan appropriate set of filters. Nuclear expression of BFA4 was observedin BT474, MDA-MB453, and MDA-MB231 breast tumour lines. No detectableexpression was found in HeLa or COS cells used as controls as well aswith secondary antibody alone.

C. Immunohistochemical Studies

To ascertain the utility of the mAbs for staining tissue sections,formalin-fixed paraffin-embedded breast cancer biopsy specimens wereobtained from the Henrietta Banting Breast Cancer Repository ofSunnybrook and Women's College Health Sciences Center (SWCHSC) inToronto, Ontario. Fifty tumour sections consisting of DCIS, invasiveductal and lobular carcinoma and mixed DCIS and invasive carcinoma werestained with 8D11 and 8A1. Antigen retrieval was performed byheat-treating the sections in a pressure cooker at 120° C. for 2.5 minin BORG buffer, pH9.5 followed by a 20-30 min cooling period withoutpressure after blocking endogenous peroxidase. The slides were washed inTris-HCl, pH 7.6 and incubated with a 1:50 dilution of affinity-purified8D11 or 8A1 mAb. After three washes in Tris buffer, the slides weretreated with Dako Envision Plus anti-mouse-HRP antibody for 30 min andwashed again three times in Tris-HCl buffer. The slides were developedwith DAB solution for ten minutes followed by counterstaining withhematoxylin, dehydration, clearing, and mounting. Arrays of normaltissues (MaxArray, Zymed Laboratories) were also stained using the samestaining protocol. Slides were scored according to the percentage ofcells and the percentage of tumour specimens staining positive. Theintensity of staining in the sections was scored as low, intermediate orhigh. A parallel set of sections from the same tumour specimens werealso stained for ER1, PR, and HER2-neu according to previously publishedmethods (Patient, et al. Curr. Opin. Genet. Dev. 12, 416-22 (2002)).

1. Expression in Normal Tissues

A striking result of the IHC analysis was the virtual absence of BFA4expression in normal tissues that were stained. Representativephotomicrographs showed a lack of any detectable BFA4 expression innormal liver, lung, colon, kidney, normal breast, and skin. Similarresults were obtained with samples from heart, stomach, spleen, liver,pancreas, small intestine, peripheral nerves, cerebral cortex,cerebellum, thyroid gland, pituitary, adrenal gland, uterus, cervix,salivary gland, and bone marrow. Some weak staining was found in thecytoplasm in a few cerebral cortical cells of brain using the 8D11.However, no staining was found with 8A1 suggesting that the weakstaining of these few cerebral cortex cells was non-specific. In bothinvasive carcinoma and DCIS patient samples, some low to intermediatelevel of BFA4 expression was seen in a proportion of the normal breastepithelial cells from these patients. The significance of this is notknown, but may indicate an abnormality in these cells in breast cancerpatients.

2. Expression in Tumor Tissues

It was observed that BFA4-stained tumour sections showing strong nuclearstaining in invasive ductal carcinomas, DCIS, mixed invasive ductalcarcinoma and DCIS. Expression of BFA4 in a RLN metastasis sample wasalso observed. The distribution of BFA4 staining in terms of percentagenuclei stained was also determined for the 50 rumours analyzed. Themajority of samples expressed BFA4 in greater than 75% of the cells inthe tumour mass. In invasive ductal carcinomas samples (n=40), 90% ofthe samples had 75% or more of the cells stained with 8D11. Similarresults were obtained using 8A1, with 84% of the samples having 75% ormore of the cells staining positive. The relationship between ER-1 andHER-2/neu status of the tumours and BFA4 expression for cases ofinvasive carcinoma and DCIS and found that BFA4 was highly expressed inER-1⁺ and ER-1⁻, as well as HER2/neu⁺ and HER2/neu⁻ tumours. Based onthis IHC analysis, it was concluded that expression of the BFA4 proteinis highly tumour specific and has a comprehensive expression throughoutthe patient population (over 90% positive) in both early and late stagesof breast cancer.

Expression of BFA4 was also determined in samples of lung carcinoma, Bcell lymphoma, T cell lymphoma, colon carcinoma, pancreatic and biliarycarcinomas, hepatocelluar carcinoma, renal cell carcinoma, and ovariancarcinoma. 8A1 and 8D11 stained a minority of B cell lymphomas andpancreatic/biliary carcinomas. 8D11 demonstrated cytoplasmic staining in9 of 18 colon carcinoma samples, 4 of 9 hepatocellular carcinomasamples, and 3 of 17 renal cell carcinoma samples. Nineteen of 20ovarian carcinoma samples were positive for nuclear staining and sixteenof 20 ovarian carcinoma samples were positive for cytoplasmic stainingusing 8D11. Sixteen of 20 ovarian carcinoma samples were positive fornuclear staining using 8A.

As shown herein, 8D11 and 8A1 may be used alone or in combination withone another to detect cancer cells of various origins. These mAbs mayalso be combined with other reagents to provide kits for identifying theexpression of BFA4.

Example 2 Kit for Identifying Cells Expressing BFA4/TRPS-1

A kit including 8D11 and/or 8A1 and optionally other componentsnecessary for using the antibodies to detect cells expressing BFA4 isprovided. The antibody may be provided in any suitable form, includingfrozen, lyophilized, or in a suitable buffer such as phosphate-bufferedsaline (PBS). In one embodiment, the 8A1 and/or 8D11 are providedindividually in purified form. In such instances, the user may choose toaffix a detectable label (i.e., biotin, FITC) to the antibody. Inanother embodiment, the kit includes 8D11 or 8A1 with a detectable label(i.e., biotin) attached. For example, in one embodiment, the kitincludes biotinylated 8A1 and/or 8D11 and an avidin-conjugated detectionreagent (i.e., secondary antibody). In another embodiment, the kitincludes a fluorescently labelled 8D11 and/or 8A1. Buffers and the likeas well as detection systems required for using any of these systems arewell-known in the art and may be prepared and utilized by the end-useror provided as a component of the kit. For example,

The kit may also include a solid support containing positive- andnegative-control protein and/or tissue samples. For example, kits forperforming spotting or western blot-type assays may include control cellor tissue lysates for use in SDS-PAGE or membranes containing pre-fixedcontrol samples with additional space for experimental samples. Kits forvisualization of BFA4 in cells on slides may include pre-formattedslides containing control cell or tissue samples with additional spacefor experimental samples.

While the present invention has been described in terms of the preferredembodiments, it is understood that variations and modifications willoccur to those skilled in the art. Therefore, it is intended that theappended claims cover all such equivalent variations that come withinthe scope of the invention as claimed.

What is claimed is:
 1. A method for determining the effectiveness of atreatment regimen for treating cancer by detecting the expression levelof an antigen in a biological sample of a patient before and aftertreatment, the antigen being reactive with the monoclonal antibody 8D11produced by the mouse hybridoma having ATCC Designation No. PTA-6852and/or a derivative thereof selected from the group consisting of anF_(ab), F_(ab2), single chain antibody, F_(v), humanized antibody, and achimeric antibody, and/or the monoclonal antibody 8A1 produced by themouse hybridoma having ATCC Designation No. PTA-6853 and/or a derivativethereof selected from the group consisting of an F_(ab), F_(ab2), singlechain antibody, F_(v), humanized antibody, and a chimeric antibody, themethod comprising: determining the level of expression of the antigen inthe biological sample obtained before treatment by detecting the bindingof the monoclonal antibody 8D11 and/or a derivative thereof and/or themonoclonal antibody 8A1 and/or a derivative thereof to components of thebiological sample; determining the level of expression of the antigen inthe biological sample obtained after treatment by detecting the bindingof the monoclonal antibody 8D11 and/or a derivative thereof and/or themonoclonal antibody 8A1 and/or a derivative thereof to components of thebiological sample; wherein: an increase in expression of the antigenafter treatment indicates the treatment is not effective; and, andecrease in expression of the antigen after treatment indicates thetreatment is effective.
 2. The method of claim 1 wherein the treatmentregimen includes a chemotherapeutic drug.
 3. The method of claim 1wherein the biological sample is a cell or tissue of the patient.
 4. Themethod of claim 3 wherein the patient is a human being.
 5. The method ofclaim 1 wherein the monoclonal antibody is monoclonal antibody 8D11produced by the mouse hybridoma having ATCC Designation No. PTA-6852and/or a derivative thereof.
 6. The method of claim 5 wherein thetreatment regimen includes a chemotherapeutic drug.
 7. The method ofclaim 5 wherein the biological sample is a cell or tissue of thepatient.
 8. The method of claim 7 wherein the patient is a human being.9. The method of claim 1 wherein the monoclonal antibody is monoclonalantibody 8D11 produced by the mouse hybridoma having ATCC DesignationNo. PTA-6852.
 10. The method of claim 9 wherein the treatment regimenincludes a chemotherapeutic drug.
 11. The method of claim 9 wherein thebiological sample is a cell or tissue of the patient.
 12. The method ofclaim 11 wherein the patient is a human being.
 13. The method of claim 1wherein the monoclonal antibody is monoclonal antibody 8A1 produced bythe mouse hybridoma having ATCC Designation No. PTA-6853 and/or aderivative thereof.
 14. The method of claim 13 wherein the treatmentregimen includes a chemotherapeutic drug.
 15. The method of claim 13wherein the biological sample is a cell or tissue of the patient. 16.The method of claim 15 wherein the patient is a human being.
 17. Themethod of claim 1 wherein the monoclonal antibody is monoclonal antibody8A1 produced by the mouse hybridoma having ATCC Designation No.PTA-6853.
 18. The method of claim 17 wherein the treatment regimenincludes a chemotherapeutic drug.
 19. The method of claim 17 wherein thebiological sample is a cell or tissue of the patient.
 20. The method ofclaim 19 wherein the patient is a human being.